Improved Current-Monitoring Method for Low Electroosmotic Flow Measurement in Modified Microchip

Current-monitoring method is a widely used approach to measure electroosmotic flow (EOF) in microchip, but low and zero EOF is difficult to be measured. In this report, the mechanism of current-monitoring method was explained with Kohlraush regulation function principle, and an improved current-monitoring method was developed for low EOF measurement with tilting microchip. Fluid flow in the channel was accelerated with the help of hydrostatic pressure generated by tilting microchip, the time of dilute solution displacing the concentrated one in channel was shortened. EOF could be calculated according to the time difference between twice experiments under two different applied voltages by tilting microchip. Low even zero EOF could be measured by this improved current-monitoring method. Three modified microchips were characterized to verify the method. EOF in microchannels modified with poly(vinyl alcohol), bovine serum albumin and myoglobin were 0.27 ± 0.05, 0.16 ± 0.05 and ?0.45 ± 0.04 × 10^?4 cm² V^?1 s^?1, respectively.     

Improved hydrostatic pressure sample injection by tilting the microchip towards the disposable miniaturized CE device

A simple method of hydrostatic pressure sample injection towards a disposable microchip CE device was developed. The liquid level in the sample reservoir was higher than that in the sample waste reservoir (SWR) by tilting microchip and hydrostatic pressure was generated, the sample was driven to pass through injection channel into SWR. After sample loading, the microchip was levelled for separation under applied high separation voltage. Effects of tilted angle, initial liquid height and injection duration on electrophoresis were investigated. With enough injection duration, the injection result was little affected by tilted angle and initial liquid heights in the reservoirs. Injection duration for obtaining a stable sample plug was mainly dependent on the tilted angle rather than the initial height of liquid. Experimental results were consistent with theoretical prediction. Fluorescence observation and electrochemical detection of dopamine and catechol were employed to verify the feasibility of tilted microchip hydrostatic pressure injection. Good reproducibility of this injection method was obtained. Because the instrumentation was simplified and no additional hardware was needed in this technology, the proposed method would be potentially useful in disposable devices.     

Low EOF rate measurement based on constant effective mobility in microchip CE

A new method for quickly determining low EOF rates (micro(EOF)) in microchip CE is described. The measurement is based on the notion that the effective mobility (micro(eff)) of an analyte is a constant in a certain BGE. The micro(eff) of an analyte is determined in a reference fast-electroosmosis microchip, and the apparent mobility (micro(app)) of the analyte can be determined in the microchip with unknown low electroosmosis, and then micro(EOF) in the low-electroosmosis microchip can be calculated according to the equation mu(EOF) = micro(app) - micro(eff). By an indirect method or other conventional methods, micro(eff) can be easily measured in the reference microchip. The proposed method is particularly useful for low-electroosmosis measurements in wall-modified microchannels.     

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.     

EOF measurement by detection of a sampling zone with end-channel amperometry in microchip CE

A simple method for EOF measurement by detection of sampling zones with end-channel amperometry in microchip CE is developed. This method is based on the principle of the Kohlrausch regulating function (KRF). A dilute electroactive ionic species is added to the BGE as a continuously eluting electrophore which is used as a probe. When a BGE-like sample at a different concentration is injected, a peak of sampling zone appears and the migration time is related to EOF. In a microchip CE with hybrid PDMS/glass channel, a cathodic EOF of the hybrid glass/PDMS microchip was measured by end-channel amperometry; the effects of sample concentration and different probes on EOF rate were discussed. The present method was applied to monitor EOF rates in glass and in PDMS microchips. There was no significant difference between the values of EOF rates measured by the present method and the current-monitoring method. Detection of nonelectroactive analytes K(+), Na(+), and Li(+) can also be accomplished by the indirect amperometric method. Hence, the effective mobility of analyte can be accurately obtained.     

Indirect amperometric measurement of electroosmotic flow rates and effective mobilities in microchip capillary electrophoresis

End-channel indirect amperometry is based on the principle of Kohlrausch regulating function (KRF). A dilute electroactive ionic species is added to the background electrolyte as a continuously eluting electrophore, which is used as probe. The probe concentration variation with the omega value of KRF in the sampling zone was described schematically in this report. Either cathodic or anodic electroosmotic flow (EOF) rates were monitored in microchip. There was no significant difference between the values of EOF rates measured by present method and current-monitoring method. Detection of electroactive and nonelectroactive analytes can also be accomplished by indirect amperometric method. Hence, the effective mobility of analytes can be accurately calculated. And the response mechanism of nonelectroactive analytes K(+), Na(+) and Li(+) in the indirect method was speculated.     

Bulk modification of polymeric microfluidic devices

The surface properties of microfluidic devices play an important role in their flow behavior. We report here on an effective control of the surface chemistry and performance of polymeric microchips through a bulk modification route during the fabrication process. The new protocol is based on modification of the bulk microchip material by tailored copolymerization of monomers during atmospheric-pressure molding. A judicious addition of a modifier to the primary monomer solution thus imparts attractive properties to the plastic microchip substrate, including significant enhancement and/or modulation of the EOF (with flow velocities comparable to those of glass), a strong pH sensitivity and high stability. Carboxy, sulfo, and amino moieties have thus been introduced (through the incorporation of methylacrylic acid, 2-sulfoethyl-methacrylate and 2-aminoethyl-methacrylate monomers, respectively). A strong increase in the electroosmotic pumping compared to the native poly(methylmethacrylate)(PMMA) microchip (ca. electroosmotic mobility increases from 2.12 to 4.30 x 10(-4) cm(2) V(-1) s(-1)) is observed using a 6% methylacrylate (MAA) modified PMMA microchip. A 3% aminoethyl modified PMMA microchip exhibits a reversal of the electroosmotic mobility (for example, -5.6 x 10(-4) cm(2) V(-1) s(-1) at pH 3.0). The effects of the modifier loading and the pH on the EOF have been investigated for the MAA-modified PMMA chips. The bulk-modified devices exhibit reproducible and stable EOF behavior. The one step fabrication/modification protocol should further facilitate the widespread production of high-performance plastic microchip devices.     

Versatile method for electroosmotic flow measurements in microchip electrophoresis

A novel versatile method for the determination of low or high electroosmotic mobility values in microdevices of variable microchannel design is presented. The electroosmotic flow (EOF) calculation is based on the difference between the apparent and effective mobilities of a reference compound. The proposed method uses microchip frontal electrophoresis for the determination of these mobilities. This requires simple monochannel microchip design and demonstrates versatile and time-saving procedure when compared to conventional current monitoring method when measuring low EOF. It has been applied successfully to the characterization of different coating procedure in glass and poly(dimethylsiloxane) microchips.     

Control of electroosmotic flow in laser-ablated and chemically modified hot imprinted poly(ethylene terephthalate glycol) microchannels

The fabrication of microchannels in poly(ethylene terephthalate glycol) (PETG) by laser ablation and the hot imprinting method is described. In addition, hot imprinted microchannels were hydrolyzed to yield additional charged organic functional groups on the imprinted surface. The charged groups are carboxylate moieties that were also used as a means for the further reaction of different chemical species on the surface of the PETG microchannels. The microchannels were characterized by fluorescence mapping and electroosmotic flow (EOF) measurements. Experimental results demonstrated that different fabrication and channel treatment protocols resulted in different EOF rates. Laser-ablated channels had similar EOF rates (5.3+/-0.3 x 10(-4) cm(2)/Vs and 5.6+/-0.4 x 10(-4) cm(2)/Vs) to hydrolyzed imprinted channels (5.1+/-0.4 x 10(-4) cm(2)/Vs), which in turn demonstrated a somewhat higher flow rate than imprinted PETG channels that were not hydrolyzed (3.5+/-0.3 x 10(-4) cm(2)/Vs). Laser-ablated channels that had been chemically modified to yield amines displayed an EOF rate of 3.38+/- 0.1 x 10(-4) cm(2)/Vs and hydrolyzed imprinted channels that had been chemically derivatized to yield amines showed an EOF rate of 2.67+/-0.6 cm(2)/Vs. These data demonstrate that surface-bound carboxylate species can be used as a template for further chemical reactions in addition to changing the EOF mobility within microchannels.     

Fabrication and performance of poly(methyl methacrylate) microfluidic chips with fiber cores

In this work, a piece of glass fiber was inserted into the channel of a poly(methyl methacrylate) (PMMA) electrophoresis microchip to enhance the electroosmotic flow (EOF) and the separation efficiency. The EOF value of the glass fiber-containing microchannel at pH 8.2 was determined to be 4.17 x 10(-4)cm2 V(-1)s(-1). The performance of the new microchip was demonstrated by its ability to separate and detect three purines coupled with end-column amperometric detection. In addition, a piece of trypsin-immobilized glass fiber was inserted into the channel of a PMMA microchip to fabricate a core-changeable microfluidic bioreactor that can be regenerated by changing the fiber. The in-channel fiber bioreactor has been coupled with matrix-assisted laser desorption ionization time-of-flight mass spectrometry for the digestion and peptide mapping of bovine serum albumin and myoglobin.     

Miniaturized two-dimensional capillary electrophoresis on a microchip for analysis of the tryptic digest of proteins

A two-dimensional capillary electrophoresis platform, combining isoelectric focusing (IEF) and capillary zone electrophoresis (CZE), was established on a microchip with the channel width and depth as 100 mum and 40 mum, respectively. With polyacrylamide as permanent coating, EOF in the microchannel, which could impair the separation, was decreased to 3.4x10(-9)m(2).V(-1).s(-1), about 1/10 of that obtained in the uncoated set-up. During the separation, peptides were first focused by IEF in the first dimensional channel, and then directly driven into the perpendicular channel by controlling the applied voltages, and separated by CZE. Effects of various experimental parameters, including the electric field strength, channel length, and injection frequency from the first to the second dimensional separation channel, were studied. Under optimized condition, the digests of BSA and proteins extracted from E. coli were separated, and a peak capacity of 540 was obtained, which was far greater than that obtained by each single dimensional separation. All these results showed the promise of multidimensional separation on a microchip for the high-throughput and high-resolution analysis of complex samples.     

Physiochemical properties of various polymer substrates and their effects on microchip electrophoresis performance

A suite of polymers were evaluated for their suitability as viable substrate materials for microchip electrophoresis applications, which were fabricated via replication technology. The relevant physiochemical properties investigated included the glass transition temperature (T(g)), UV-vis absorption properties, autofluorescence levels, electroosmotic flow (EOF) and hydrophobicity/hydrophilicity as determined by sessile water contact angle measurements. These physiochemical properties were used as a guide to select the proper substrate material for the intended microchip electrophoretic application. The T(g) of these polymers provided a guide for optimizing embossing parameters to minimize replication errors (REs), which were evaluated from surface profilometer traces. RE values ranged from 0.4 to 13.6% for the polymers polycarbonate (PC) and low-density polyethylene (LDPE), respectively. The absorption spectra and autofluorescence levels of the polymers were also measured at several different wavelengths. In terms of optical clarity (low absorption losses and small autofluorescence levels), poly(methyl methacrylate), PMMA (clear acrylic), provided ideal characteristics with autofluorescence levels comparable to glass at excitation wavelengths that ranged from 488-780 nm. Contact angle measurements showed a maximum (i.e., high degree of hydrophobicity) for polypropylene (PP), with an average contact angle of 104 degrees +/-3 degrees and a minimum exhibited by gray acrylic, G-PMMA, with an average contact angle of 27 degrees +/-2 degrees. The EOF was also measured for thermally assembled chips both before and after treatment with bovine serum albumin (BSA). The electrophoretic separation of a mixture of dye-labeled proteins including; carbonic anhydrase, phosphorylase B, beta-galactosidase, and myosin, was performed on four different polymer microchips using laser-induced fluorescence (LIF) excitation at 632.8 nm. A maximum average resolution of 5.04 for several peak pairs was found with an efficiency of 6.68 x 10(4) plates for myosin obtained using a BSA-treated PETG microchip.     

Circular Hydraulic Jumps Triggered by Boundary Layer Separation

When a high-flow-rate circular jet impinges vertically on a horizontal plane, it flows out radially and then undergoes a distinctive hydraulic jump on the plane because of boundary layer separation induced by hydrostatic back pressure. The jump radius is shown to be 0.37 a Re(1/3) Lambda(-1/8), where Lambda=(ga(3)/nu(2)) Re(-7/3) is a modified Froude number, Re=(Q/anu) is the jet Reynolds number, a is the jet radius, and Q the liquid flow rate, which is favorably compared to experimental data in the limit of small Lambda. When Lambda exceeds 3.0x10(-4) at low flow rates, the jump radius decreases below a minimum in the film depth and our experiments detect a different jump mechanism that may be triggered by capillary pressure rather than hydrostatic pressure. Copyright 2001 Academic Press.     

Pressure generation at the junction of two microchannels with different depths

In this study, we report the design of a microchip‐based hydraulic pump that comprises three glass conduits arranged in a T‐geometry, one of which has a 2 mm long segment shallower (0.5–3 μm in depth) than the remaining 15 μm deep microfluidic network. Upon application of an electric field across this microchannel junction, a mismatch in EOF rate is introduced due to a differential in the fluid conductivity across the deep and shallow segments. Using the reported micropump, pressure‐driven velocities up to 3.2 mm/s have been generated in a 15 μm deep separation channel for an applied voltage of 1.75 kV allowing us to operate under separation conditions that yield the minimum plate height. Moreover, we have shown that this flow velocity can be maximized by optimizing the depth in the shallow region of the T‐geometry. Interestingly however, a simple theory accounting for fluid conductivity differences across microchannels of different depths significantly underestimates the pressure‐driven velocities observed in our experiments. The Taylor dispersion coefficient in our system on the other hand compares well with the theoretical predictions reported in the literature. Finally, the functionality of our device has been demonstrated by implementing a reverse‐phase chromatographic separation that was driven by the pressure‐driven flow generated on‐chip.     

Combination of large‐volume sample stacking with an electroosmotic flow pump with field‐amplified sample injection on cross‐channel chips

A combination of two online sample concentration techniques, large‐volume sample stacking with an electroosmotic flow (EOF) pump (LVSEP) and field‐amplified sample injection (FASI), was investigated in microchip electrophoresis (MCE) to achieve highly sensitive analysis. By applying reversed‐polarity voltages on a cross‐channel microchip, anionic analytes injected throughout a microchannel were first concentrated on the basis of LVSEP, followed by the electrokinetic stacking injection of the analytes from a sample reservoir by the FASI mechanism. As well as the voltage application, a pressure was also applied to the sample reservoir in LVSEP‐FASI. The applied pressure generated a counter‐flow against the EOF to reduce the migration velocity of the stacked analytes, especially around the cross section of the microchannel, which facilitated the FASI concentration. At the hydrodynamic pressure of 15 Pa, 4520‐fold sensitivity increase was obtained in the LVSEP‐FASI analysis of a standard dye, which was 33‐times higher than that obtained with a normal LVSEP. Furthermore, the use of the sharper channel was effective for enhancing the sensitivity, e.g., 29 100‐fold sensitivity increase was achieved with the 75‐μm wide channel. The developed method was applied to the chiral analysis of amino acids in MCE, resulting in the sensitivity enhancement factor of 2920 for the separated d ‐leucine.     

Further improvement of hydrostatic pressure sample injection for microchip electrophoresis

Hydrostatic pressure sample injection method is able to minimize the number of electrodes needed for a microchip electrophoresis process; however, it neither can be applied for electrophoretic DNA sizing, nor can be implemented on the widely used single-cross microchip. This paper presents an injector design that makes the hydrostatic pressure sample injection method suitable for DNA sizing. By introducing an assistant channel into the normal double-cross injector, a rugged DNA sample plug suitable for sizing can be successfully formed within the cross area during the sample loading. This paper also demonstrates that the hydrostatic pressure sample injection can be performed in the single-cross microchip by controlling the radial position of the detection point in the separation channel. Rhodamine 123 and its derivative as model sample were successfully separated.     

On-chip pumping for pressure mobilization of the focused zones following microchip isoelectric focusing

Isoelectric focusing (IEF), traditionally accomplished in slab or tube gels, has also been performed extensively in capillary and, more recently, in microchip formats. IEF separations performed in microchips typically use electroosmotic flow (EOF) or chemical treatment to mobilize the focused zones past the detection point. This report describes the development and optimization of a microchip IEF method in a hybrid PDMS-glass device capable of controlling the mobilization of the focused zones past the detector using on-chip diaphragm pumping. The microchip design consisted of a glass fluid layer (separation channels), a PDMS layer and a glass valve layer (pressure connections and valve seats). Pressure mobilization was achieved on-chip using a diaphragm pump consisting of a series of reversible elastomeric valves, where a central diaphragm valve determined the volume of solution displaced while the gate valves on either side imparted directionality. The pumping rate could be adjusted to control the mobilization flow rate by varying the actuation times and pressure applied to the PDMS to actuate the valves. In order to compare the separation obtained using the chip with that obtained in a capillary, a serpentine channel design was used to match the separation length of the capillary, thereby evaluating the effect of diaphragm pumping itself on the overall separation quality. The optimized mIEF method was applied to the separation of labeled amino acids.     

Gravity-induced convective flow in microfluidic systems: electrochemical characterization and application to enzyme-linked immunosorbent assay tests

A way of using gravity flow to induce a linear convection within a microfluidic system is presented. It is shown and mathematically supported that tilting a 1 cm long covered microchannel is enough to generate flow rates up to 1000 nL.min(-1), which represents a linear velocity of 2.4 mm.s(-1). This paper also presents a method to monitor the microfluidic events occurring in a covered microchannel when a difference of pressure is applied to force a solution to flow in said covered microchannel, thanks to electrodes inserted in the microfluidic device. Gravity-induced flow monitored electrochemically is applied to the performance of a parallel-microchannel enzyme-linked immunosorbent assay (ELISA) of the thyroid-stimulating hormone (TSH) with electrochemical detection. A simple method for generating and monitoring fluid flows is described, which can, for instance, be used for controlling parallel assays in microsystems.     

A circular ferrofluid driven microchip for rapid polymerase chain reaction

In the past few years, much attention has been paid to the development of miniaturized polymerase chain reaction (PCR) devices. After a continuous flow (CF) PCR chip was introduced, several CFPCR systems employing various pumping mechanisms were reported. However, the use of pumps increases cost and imposes a high requirement on microchip bonding integrity due to the application of high pressure. Other significant limitations of CFPCR devices include the large footprint of the microchip and the fixed cycle number which is dictated by the channel layout. In this paper, we present a novel circular close-loop ferrofluid driven microchip for rapid PCR. A small ferrofluid plug, containing sub-domain magnetic particles in a liquid carrier, is driven by an external magnet along the circular microchannel, which in turn propels the PCR mixture through three temperature zones. Amplification of a 500 bp lambda DNA fragment has been demonstrated on the polymethyl methacrylate (PMMA) PCR microchip fabricated by CO(2) laser ablation and bonded by a low pressure, high temperature technique. Successful PCR was achieved in less than 4 min. Effects of cycle number and cycle time on PCR products were investigated. Using a magnet as the actuator eliminates the need for expensive pumps and provides advantages of low cost, small power consumption, low requirement on bonding strength and flexible number of PCR cycles. Furthermore, the microchip has a much simpler design and smaller footprint compared to the rectangular serpentine CFPCR devices. To demonstrate its application in forensics, a 16-loci short tandem repeat (STR) sample was successfully amplified using the PCR microchip.     

Unsteady electroosmosis in a microchannel with Poisson-Boltzmann charge distribution

The present study is concerned with unsteady electroosmotic flow (EOF) in a microchannel with the electric charge distribution described by the Poisson-Boltzmann (PB) equation. The nonlinear PB equation is solved by a systematic perturbation with respect to the parameter λ which measures the strength of the wall zeta potential relative to the thermal potential. In the small λ limits (λ<1), we recover the linearized PB equation - the Debye-Hückel approximation. The solutions obtained by using only three terms in the perturbation series are shown to be accurate with errors <1% for λ up to 2. The accurate solution to the PB equation is then used to solve the electrokinetic fluid transport equation for two types of unsteady flow: transient flow driven by a suddenly applied voltage and oscillatory flow driven by a time-harmonic voltage. The solution for the transient flow has important implications on EOF as an effective means for transporting electrolytes in microchannels with various electrokinetic widths. On the other hand, the solution for the oscillatory flow is shown to have important physical implications on EOF in mixing electrolytes in terms of the amplitude and phase of the resulting time-harmonic EOF rate, which depends on the applied frequency and the electrokinetic width of the microchannel as well as on the parameter λ.