Integration of continuous-flow sampling with microchip electrophoresis using poly(dimethylsiloxane)-based valves in a reversibly sealed device

Here we describe a reversibly sealed microchip device that incorporates poly(dimethylsiloxane) (PDMS)-based valves for the rapid injection of analytes from a continuously flowing stream into a channel network for analysis with microchip electrophoresis. The microchip was reversibly sealed to a PDMS-coated glass substrate and microbore tubing was used for the introduction of gas and fluids to the microchip device. Two pneumatic valves were incorporated into the design and actuated on the order of hundreds of milliseconds, allowing analyte from a continuously flowing sampling stream to be injected into an electrophoresis separation channel. The device was characterized in terms of the valve actuation time and pushback voltage. It was also found that the addition of sodium dodecyl sulfate (SDS) to the buffer system greatly increased the reproducibility of the injection scheme and enabled the analysis of amino acids derivatized with naphthalene-2,3-dicarboxaldehyde/cyanide. Results from continuous injections of a 0.39 nL fluorescein plug into the optimized system showed that the injection process was reproducible (RSD of 0.7%, n = 10). Studies also showed that the device was capable of monitoring off-chip changes in concentration with a device lag time of 90 s. Finally, the ability of the device to rapidly monitor on-chip concentration changes was demonstrated by continually sampling from an analyte plug that was derivatized upstream from the electrophoresis/continuous flow interface. A reversibly sealed device of this type will be useful for the continuous monitoring and analysis of processes that occur either off-chip (such as microdialysis sampling) or on-chip from other integrated functions.     

芯片毛细管电泳-激光诱导荧光-电荷耦合器件检测系统

采用自组建的芯片毛细管电泳－激光诱导荧光－电荷耦合器件（CCD）检测系统在数十秒内满意地分离了曙红和荧光素。设计了一种进样、分 离电路,可以有效地消除进样通道的样品溶液向分离通道的渗漏。解决了由这种渗漏所引起的电泳峰变宽、拖尾等问题。提高了芯片毛细管电泳的分辨率和分离效率。     

超高速平板通道毛细管电泳

超高速平板通道毛细管电泳是９０年代发展的一种秒级分离的新颖技术。应用现代微电子光刻技术将化学反应。进样、分离和检测等组合在数厘米玻片上。实现分离分析的小型化、集成化、一体化和自动化。     

A compactly integrated laser-induced fluorescence detector for microchip electrophoresis

A simple and easy-to-use integrated laser-induced fluorescence detector for microchip electrophoresis was constructed and evaluated. The fluid channels and optical fiber channels in the glass microchip were fabricated using standard photolithographic techniques and wet chemical etching. A 473 nm diode-pumped laser was used as the excitation source, and the collimation and collection optics and mirrors were discarded by using a multimode optical fiber to couple the excitation light straight into the microchannel and placing the microchip directly on the top of the photomultiplier tube. A combination of filter systems was incorporated into a poly(dimethylsiloxane) layer, which was reversibly sealed to the bottom of the microchip to eliminate the scattering excitation light reaching to the photomultiplier tube. Fluorescein/calcein samples were taken as model analytes to evaluate the performance with respect to design factors. The detection limits were 0.05 microM for fluorescein and 0.18 microM for calcein, respectively. The suitability of this simple detector for fluorescence detection was demonstrated by baseline separation of fluorescein isothiocyanate (FITC)-labeled arginine, phenylalanine, and glycine and FITC within 30 s at separation length of 3.8 cm and electrical field strength of 600 V/cm.     

Electrophoretically mediated microanalysis of beta-galactosidase on microchips.

Electrophoretically mediated microanalysis (EMMA) is a method of accomplishing chemical analyses, typically in an open-tubular capillary, due to the difference in the electrophoretic mobility between the particular reagents. This work reports on combining this technique onto microfabricated systems. Two methods of this technique were applied, constant potential and zero potential EMMA onto chips. A dosage response curve was run using this constant potential mode that resulted in a linear response over three orders of substrate concentration magnitude. The chemical system used here is beta-galactosidase (beta-Gal) as the enzyme and fluorescein mono-beta-D-galactopyranoside (FMG) as the substrate. The zero potential mode was used to amplify product turnover using various incubation times. Using this technique and a 10 min incubation, approximately 40000 enzyme molecules could be detected. The zero potential mode is also used in conjunction with an internal standard to show how one can quantitate using this method. The power and ease of utility of this technique is described.     

A rigid poly(dimethylsiloxane) sandwich electrophoresis microchip based on thin-casting method

We present a novel concept of glass/poly(dimethylsiloxane) (PDMS)/glass sandwich microchip and developed a thin-casting method for fabrication. Unlike the previously reported casting method for fabricating PDMS microchip, several drops of PDMS prepolymer were first added on the silanizing SU-8 master, then another glass plate was placed over the prepolymer as a cover plate, and formed a glass plate/PDMS prepolymer/SU-8 master sandwich mode. In order to form a thin PDMS membrane, a weight was placed on the glass plate. After the whole sandwich mode was cured at 80 degrees C for 30 min, the SU-8 master was easily peeled and the master microstructures were completely transferred to the PDMS membrane which was tightly stuck to the glass plate. The microchip was subsequently assembled by reversible sealing with the glass cover plate. We found that this PDMS sandwich microchip using the thin-casting method could withstand internal pressures of >150 kPa, more than 5 times higher than that of the PDMS hybrid microchip with reversible sealing. In addition, it shows an excellent heat-dissipating property and provides a user-friendly rigid interface just like a glass microchip, which facilitates manipulation of the microchip and fix tubing. As an application, PDMS sandwich microchips were tested in the capillary electrophoresis separation of fluorescein isothiocyanate-labeled amino acids.     

A thin cover glass chip for contactless conductivity detection in microchip capillary electrophoresis

A microfabricated thin glass chip for contactless conductivity detection in chip capillary electrophoresis is presented in this contribution. Injection and separation channels were photolithographed and chemically etched on the surface of substrate glass, which was bonded with a thin cover glass (100 μm) to construct a new microchip. The chip was placed over an independent contactless electrode plate. Owing to the thinness between channel and electrodes, comparatively low excitation voltage (20–110 V in V_p–p) and frequency (40–65 kHz) were suitable, and favorable signal could be obtained. This microchip capillary electrophoresis device was used in separation and detection of inorganic ions, amino acids and alkaloids in amoorcorn tree bark and golden thread in different buffer solutions. The detection limit of potassium ion was down to 10 μmol/L. The advantages of this microchip system exist in the relative independence between the microchip and the detection electrodes. It is convenient to the replacement of chip and other operations. Detection in different position of the channel would also be available.     

Microchannel electrophoretic separation of biogenic amines by micellar electrokinetic chromatography

Fast, efficient separation of most common biogenic amines was successfully performed on a glass microchip capillary electrophoresis device. The amines putrescine, histamine, tyramine, cadaverine, phenethylamine, tryptamine, spermidine and spermine were derivatized prior to fluorescence detection with fluorescein isothiocyanate. Separation was carried out using a channel length of 28 mm, a cross section of 50 x 8 microm, and a field strength of 600 V/cm. After optimization of buffer electrolyte conditions (120 mM boric acid, pH 9.4, modified with 40 mM SDS), fluorescein thiocarbamyl amine derivatives were successfully resolved. Analysis time was as short as 75 s. Determination of the biogenic amines was achieved in soy sauce samples.     

High-throughput characterization for organic pollutants in environmental waters using a capillary electrophoresis chip.

To evaluate organic pollution in water, we did preliminarily studies on high-throughput characterization of organic pollution in water using microchip-based capillary electrophoresis (CE) with laseer-induced fluorescence (LIF) detection. The applied voltage was investigated to control the gated valve injection and CE separation for conventional cross type microchips using a self-made personal computer (PC)-based controller as the voltage supply. We obtained high-throughput data for the reproducible separation of fluorescein isothiocyanate (FITC)-labeled river-water samples using a zwitter-ion based buffer solution to avoid adsorption of the labeled sample onto the channel of a microchip made from quartz glass. We used real samples from the Hino River that flows into Lake Biwa, from ten sampling points and obtained several reproducible peaks in different separation patterns for each sample within 2 min. We successfully demonstrated high-throughput characterization of dissolved organic carbon (DOC) in environmental water using the microchip.     

Frontal analysis on a microchip

Conventional microchip applications involving capillary electrophoresis (CE) typically inject a sample along one channel and use an intersection of two channels to define the sample plug--the portion of sample to be analysed along a second channel. In contrast to this method of zone separation, frontal analysis proceeds by injecting sample continuously into a single channel or column. Frontal analysis is more common in macroscopic procedures but there are benefits in sensitivity and device density to its application to electrophoresis on microchips. This work compares conventional microchip zone analysis with frontal analysis in the separation of PCR products. Although we detect on the order of 5000 fluorophores with a compact instrument using the zone separation CE method, we found a several-fold increase in the effective signal-to-noise ratio by using a frontal analysis method. By removing the need for additional channels and reservoirs the frontal method would allow device densities to be significantly increased, potentially improving the cost-effectiveness of microchip analyses in applications such as medical diagnostics.     

Fast haptoglobin phenotyping based on microchip electrophoresis

A new and fast method for haptoglobin phenotyping was developed based on microchip electrophoresis with laser induced fluorescence detection. Haptoglobin phenotypes 1-1 and 2-2 were labeled with fluorescein isothiocyanate. The analyses were performed on glass microchip which was simply treated with sodium dodecyl sulfate. After the optimization of the separation conditions, Hp 1-1 and Hp 2-2 could be differentiated in 150 s and the detection limits for Hp 1-1 and Hp 2-2 were 0.39 and 0.62 μg/mL, respectively. Finally, the method was applied to human serum samples from healthy people and liver cancer patients. A decrease in Hp concentration for liver cancer patients was confirmed. Featuring high efficiency, speed, simplicity, the method reveals great potentials for the diagnosis of diseases and proteome research.     

Coupling a microchip with electrospray ionization quadrupole time-of-flight mass spectrometer for peptide separation and identification

The present study reports a simple method of coupling a glass microchip to an electrospray ionization (ESI) quadrupole time-of-flight mass spectrometer (QTOF-MS) for separation and identification of peptides. A sheath-flow electrospray interface was constructed based on attaching a short fused-silica capillary to the microchip. The dead volume at the interface was effectively reduced by wet etching an approximate flat-bottom capillary insertion channel coaxial to the end of separation microchannel and using a wire-controlled epoxy-blocking attachment method. The makeup liquid and neb gas were coaxially pumped through two stainless-steel tees to maintain a stable and efficient electrospray. The coupled microchip/ESI-QTOF-MS system was successfully used to carry out electrophoresis separation of peptides and ESI-QTOF-MS identification.     

High-throughput enzyme assay on a multichannel microchip using optically gated sample introduction

To meet the requirements for high-throughput screening for drug discovery research, it is very important to develop techniques with the ability of performing multiple enzyme assays simultaneously. Using optically gated sample introduction on a multichannel microchip, multiple enzyme assays have been demonstrated in four parallel channels. The hydrolysis of fluorescein mono-beta-D-galactopyranoside by beta-galactosidase and the inhibition of this reaction by the competitive inhibitor phenylethyl beta-D-thiogalactoside were initially studied to determine the effect of system movement using the voice coil actuator on the enzyme assay reaction. The results from these two studies are consistent with the results from the assay using a single-channel microchip, and they demonstrate that the system using optically gated sample introduction on multichannel microchip can be used to perform multiple enzyme assays. Three unique enzyme assays were also performed in different channels, which show this technique could be competitive for high-throughput screening in drug discovery with other traditional techniques.     

Property of ionic liquid in electrophoresis and its application in chiral separation on microchips

The aqueous solution of a kind of room-temperature ionic liquids (RTILs), 1-ethyl-3-methylimidazolium-tetrafluoroborate (1E-3MI-TFB), demonstrated its exclusive electroosmotic property in microchip electrophoresis. It was applied as the working electrolyte for chiral separation in glass microchip electrophoresis. Compared with boric acid buffer, 1E-3MI-TFB aqueous solution exhibited a broader separation window for enantiomers of dipeptides. Then the influences of chiral selector, pH and concentration on efficiency of chiral separation were discussed in detail. The unique mechanism of the generation of EOF was explored in a glass microchannel using 1E-3MI-TFB aqueous solution as working electrolyte. A possible status of 1E-3MI cation in water was suggested at the first time, which facilitated the explanation of EOF and its characteristics in glass microchannel. Additionally, microchip electrophoresis using 1E-3MI-TFB aqueous solution was successfully applied to the chiral separation of complex enantiomers of dipeptides. RTILs aqueous solution, as the electrolyte for the separation of complicated optical isomers, could lead to a revolution in the analytical methods of chiral or conformational analysis for biomolecules.     

A simple PDMS-based electro-fluidic interface for microchip electrophoretic separations

High voltage electrodes for electrophoresis have been integrated into a polymer layer that can be reversibly bound to glass microchips for electrophoretic separations. By using the liquid precursor to the polymer polydimethylsiloxane (PDMS), platinum electrodes and reservoirs can be positioned prior to solidification, providing a simple and flexible method for electrode interface construction. Field strengths up to 875 V cm(-1) over an 8 cm separation channel can be applied to the system without any loss in performance of the interface. The interface can function as an electro-fluidic interface between the high voltage power supply and the separation channel and, when reversibly sealed to an etched glass plate, functions as a cover plate establishing a hybrid PDMS-glass microchip in which the electrodes are directly integrated onto the device. The versatility of this approach is not only demonstrated by separating DNA fragments in a novel buffer sieving matrix, but also with the molecular diagnostic analysis of a variety of DNA samples for Duschenne Muscular Dystrophy and cytomegalovirus (CMV) infection, using both microchip interface configurations.     

集成化液膜萃取-反萃取-毛细管电泳联用芯片

我们设计并制作了集成有支持液膜萃取-反萃取试样预处理的毛细管电泳(SLMEBE-CCE)微流控芯片. 分别以荧光素钠和丁基罗丹明B作为模型待测物和共存物, 在该芯片上进行了在线试样预处理与毛细管电泳联用的初步实验.     

Protein separation using free‐flow electrophoresis microchip etched in a single step

A one‐step etching method was developed to fabricate glass free‐flow electrophoresis microchips with a rectangle separation microchamber (42 mm‐long, 23 mm‐wide and 28 μm‐deep), in which two glass bridges (0.5 mm‐wide) were made simultaneously to prevent bubbles formed by electrolysis near the Pt electrode from entering the separation chamber. By microchip free‐flow zone electrophoresis, with 200 V voltage applied, the baseline separation of three FITC labeled proteins, ribonuclease B, myoglobin and β‐lactoglobulin, was achieved, with resolution over 1.78. Furthermore, with 2.5 mM Na₂SO₄ added into the electrode buffer to form higher electrical field strength across separation microchamber than electrode compartments, similar resolution of samples was achieved with the applied voltage decreased to 75 V, which could obviously decrease Joule heat during continuous separation. All these results demonstrate that the free‐flow electrophoresis microchip fabricated by one‐step etching method is suitable for the continuous separation of proteins, which might become an effective pre‐fractionation method for proteome study.     

A rapid enzyme assay for<Emphasis Type="Italic"> β</Emphasis>-galactosidase using optically gated sample introduction on a microfabricated chip

The ability to perform enzyme assays on microchips is demonstrated using optically gated sample introduction. The hydrolysis of fluorescein mono--d-galactopyranoside (FMG) by -d-galactosidase (-Gal) is continuously monitored using a microchip for 5 to 10 min. The outcome of the reaction was analyzed by performing serial on-chip separations of fluorescent substrate, FMG, and product, fluorescein. Kinetic information about -Gal has been successfully obtained by varying the concentration of FMG. -Gal enzymes from two different sources including bovine liver and E. coli., have been examined and compared to each other and to results obtained using traditional assay methods. In addition, the competitive inhibition of -Gal by phenylethyl -d-thiogalactoside (PETG) and -lactose has been studied using this technique. PETG is found to have higher inhibition than lactose in the hydrolysis. This separation-based enzyme assay technique avoids the possible fluorescence interference between FMG and fluorescein, which is a problem with the traditional plate assay method. Additionally, the amount of the enzyme and substrate required with this technique is at least four orders of magnitude lower than the traditional plate assay method. By using optically gated sample introduction, microchips allow continuous serial injections and separations without any potential switch, thus making this technique ideal as a sensor for enzyme assays. This technique should therefore be valuable for high-throughput screening in the drug discovery industry.     

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.     

Electrokinetic characterization of hybrid NOA 81-glass microchips: Application to protein microchip electrophoresis with indirect fluorescence detection

A low-cost and straightforward hybrid NOA (Norland optical adhesive) 81-glass microchip electrophoresis device was designed and developed for protein separation using indirect fluorescence detection. This new microchip was first characterized in terms of surface charge density via electroosmotic mobility measurement and stability over time. A systematic determination of the electroosmotic mobility (μ_eo) over a wide pH range (2–10) and at various ionic strengths (20–50 mM) was developed for the first time via the neutral marker approach in an original simple frontal methodology. The evolution of μ_eo was proved consistent with the silanol and thiol functions arising from the glass and the NOA materials, respectively. The repeatability and reproducibility of the measurements on different microchips (RSD < 14%) and within 15 days (less than 5% decrease) were successfully demonstrated. The microchip was then applied for the efficient electrophoretic separation of proteins in a zonal mode coupled with indirect fluorescence detection, which is, to our knowledge, the first proof of concept of capillary zone electrophoresis in this hybrid microsystem.