硅基电泳芯片非接触电导检测器

研制了一种集成于硅基电泳芯片分离沟道末端侧壁的新型二电极非接触电导检测器.讨论了影响电导检测响应灵敏度的相关因素;采用MEMS分析软件及等效电路模拟仿真,确定了检测器的相关参数,电极长度为550μm,宽度为15μm,间距为80μm,绝缘层厚度为1μm,电导检测工作频率为300 kHz.在加工技术中,选用SOI(sili-con on insulator)基片制作十字形微沟道及集成电导检测电极,采用深刻蚀和隔离技术使检测电极被完全隔离成孤岛,利用硼掺杂技术在分离沟道末端侧壁形成立体电极,获得了集成非接触电导检测电极的硅基电泳芯片.在外加Vpp为10 V、工作频率为300 kHz的正弦波激励下,进行了Na+无机阳离子浓度梯度实验以及Na+和Li+混合无机阳离子的电泳分离检测.结果表明,Na+浓度在1×10-9～1×10-4 mol/L范围内,电导响应信号随着离子浓度的增加而增大,检出限达到1×10-9 mol/L;Na+和Li+混合无机阳离子的分离度达到2.0,实现基线分离.     

毛细管电泳非接触电导检测器的研制

非接触电导检测法已经成为毛细管电泳芯片中一种通用的物质分离检测方法．本文提出了一种微型化的电容耦合非接触电导分析系统,采用新颖的三明治电极结构减小了电极之间的寄生电容．这种电极结构能够有效地减小电极的长度,克服了非接触电导检测中电极易于折断的缺点．当采用电极宽度1mm、电极间距1mm、MES／His缓冲溶液浓度为20mmol／L和激励电压20Vpp、90kHz的条件时,检测器具有最好的分离效果．在最佳的分离效果,实现了1mmol／L的K^＋和Mg^2＋混合无机阳离子的分离检测．     

侧壁四电极电容耦合非接触电导检测器(英文)

研制了一种集成于硅基电泳芯片分离沟道末端侧壁的新型四电极电容耦合非接触电导检测器.研究了该电导检测器的等效模型,对等效电路模型中的参数进行了公式推导,并讨论了影响电导检测响应灵敏度的相关因素.采用深刻蚀及离子注入加工技术制得了用于电导检测的立体电极.制作了基于锁相放大原理的信号处理电路,对该电导检测的频率响应及灵敏度进行了测试分析.实验结果表明,当激励信号频率为300 kHz时,该电导检测器具有最佳线性度;不同浓度Na+溶液响应电压差值为5 mV;检测限达到10-8mol/L;且成功实现了Na+和Li+混合无机阳离子的电泳分离在线检测.     

Contact conductivity detection in poly(methyl methacrylate)-based microfluidic devices for analysis of mono- and polyanionic molecules

An on-column contact conductivity detector was developed for the analysis of various mono- and polyanionic compounds separated by electrophoresis chips fabricated in poly(methyl methacrylate) (PMMA) using hot embossing techniques from Ni electroforms. The detector consisted of a pair of Pt wires (127 microm diameter) with an end-to-end spacing of approximately 20 microm and situated within the fluidic channel. The waveform applied to the electrode pair was a bipolar pulse with a frequency of 5.0 kHz and was used to reduce the charging current from measurement so that the current recorded at the end of one pulse is more representative of the solution conductivity. Using the detector, separations of amino acids, peptides, proteins, and oligonucleotides were demonstrated. For the amino acids and peptides, free-solution zone electrophoresis was performed. A calibration plot for the amino acid alanine was found to be linear from approximately 10 to 100 nM in a carrier electrolyte consisting of 10 mM triethylamonium acetate. The concentration detection limit was found to be 8.0 nM, with the corresponding mass detection limit equal to 3.4 amol (injection volume = 425 pL). The protein separations with conductivity detection were performed using MEKC, in which the carrier electrolyte contained the anionic surfactant sodium dodecyl sulfate (SDS) above its cmc. Near baseline resolution was achieved in the PMMA microchip for a solution containing 8 different proteins. In the case of the DNA fragments, capillary electrochromatography was used with a C18-modified PMMA chip and a carrier electrolyte containing an ion-pairing agent.     

Movable contactless-conductivity detector for microchip capillary electrophoresis

A new movable contactless-conductivity detection system for microchip capillary electrophoresis is introduced. Such a versatile system relies on positioning the detector at different points along the separation channel via "sliding" the electrode holder. The new movable microchip detection system offers distinct improvements compared to common fixed-location conductivity detectors. For example, placing the detector at different locations along the microchannel offers useful insights into the separation process. Three-dimensional plots of resolution/channel length/separation voltage can be used for optimizing the separation process and selecting the analysis time. The system enables rapid switching between "total" (unresolved) and "individual" (resolved/fingerprint) signals on the basis of placing the detector at the beginning and end of the separation channel, respectively. By moving the detector to a shorter effective separation length, after eluting fast-migrating ions, shorter analysis times can be achieved (through faster detection of late-eluting analytes). These and other improvements in the analytical performance and insights into the separation process are illustrated in connection with the detection of low-energy ionic explosives and nerve agent degradation products.     

Nonaqueous electrophoresis microchip separations: conductivity detection in UV-absorbing solvents

The use of organic solvents in microfabricated capillary electrophoresis (CE) devices is demonstrated in connection with the separation of aliphatic amines in pure dimethylformamide, dimethylacetamide, dimethyl sulfoxide, or propylene carbonate media. Contactless conductivity detection is employed for monitoring the separated solutes in these UV-absorbing solvents. The effect of the physicochemical properties of the organic solvent upon the migration behavior is investigated. The apparent mobility increases nearly linearly with the reciprocal of the solvent viscosity, while the electroosmotic mobility increases in a linear fashion with the dielectric constant/ viscosity ratio. Some deviation from theoretical predictions is observed using propylene carbonate. The nonaqueous CE microchip offers high separation efficiency, reflected in plate numbers ranging from 93,680 to 127,680, using a separation voltage of +3,000 V, a dimethylformamide medium, and a contactless conductivity detection. Experimental parameters affecting the analytical performance of the nonaqueous CE/conductivity microchip are examined. Calibration and precision experiments indicate a linear and reproducible response. Such use of organic solvents can benefit microchip separations through extended scope (toward nonpolar solutes) and tunable selectivity.     

Multichannel microchip electrophoresis device fabricated in polycarbonate with an integrated contact conductivity sensor array

A 16-channel microfluidic chip with an integrated contact conductivity sensor array is presented. The microfluidic network consisted of 16 separation channels that were hot-embossed into polycarbonate (PC) using a high-precision micromilled metal master. All channels were 40 microm deep and 60 microm wide with an effective separation length of 40 mm. A gold (Au) sensor array was lithographically patterned onto a PC cover plate and assembled to the fluidic chip via thermal bonding in such a way that a pair of Au microelectrodes (60 microm wide with a 5 microm spacing) was incorporated into each of the 16 channels and served as independent contact conductivity detectors. The spacing between the corresponding fluidic reservoirs for each separation channel was set to 9 mm, which allowed for loading samples and buffers to all 40 reservoirs situated on the microchip in only five pipetting steps using an 8-channel pipettor. A printed circuit board (PCB) with platinum (Pt) wires was used to distribute the electrophoresis high-voltage to all reservoirs situated on the fluidic chip. Another PCB was used for collecting the conductivity signals from the patterned Au microelectrodes. The device performance was evaluated using microchip capillary zone electrophoresis (mu-CZE) of amino acid, peptide, and protein mixtures as well as oligonucleotides that were separated via microchip capillary electrochromatography (mu-CEC). The separations were performed with an electric field (E) of 90 V/cm and were completed in less than 4 min in all cases. The conductivity detection was carried out using a bipolar pulse voltage waveform with a pulse amplitude of +/-0.6 V and a frequency of 6.0 kHz. The conductivity sensor array concentration limit of detection (SNR = 3) was determined to be 7.1 microM for alanine. The separation efficiency was found to be 6.4 x 10(4), 2.0 x 10(3), 4.8 x 10(3), and 3.4 x 10(2) plates for the mu-CEC of the oligonucleotides and mu-CZE of the amino acids, peptides, and proteins, respectively, with an average channel-to-channel migration time reproducibility of 2.8%. The average resolution obtained for mu-CEC of the oligonucleotides and mu-CZE of the amino acids, peptides, and proteins was 4.6, 1.0, 0.9, and 1.0, respectively. To the best of our knowledge, this report is the first to describe a multichannel microchip electrophoresis device with integrated contact conductivity sensor array.     

Three-electrode electrochemical detector and platinum film decoupler integrated with a capillary electrophoresis microchip for amperometric detection

This article demonstrates that a three-electrode electrochemical (EC) detector and an electric decoupler could be fabricated in the same glass chip and integrated with an O2-plasma-treated PDMS layer using microfabrication techniques to form the capillary electrophoresis (CE) microchip. The platinized decoupler could mostly decouple the electrochemical detection circuit from the interference of an separation electric field in 10 mM 2-(N-morpholino)ethanesulfonic acid (MES, pH 6.5) solution. The baseline offset of background current recorded from the working electrode with and without application of a separation electric field was maintained at less than 0.05 pA in 10 mM MES. In addition, the platinized pseudoreference electrode was demonstrated to offer a stable potential in electrochemical detection. As a consequence, the limit of detection of dopamine was 0.125 microM at a S/N = 4. The responses for dopamine to different concentrations were found to be linear between 0.25 and 50 microM with a correlation coefficient of 0.9974 and a sensitivity of 11.76 pA/microM. The totally integrated CE-EC microchip should be able to fulfill the ideal of miniaturization and commercialization.     

Improving the compatibility of contact conductivity detection with microchip electrophoresis using a bubble cell

A new approach for improving the compatibility between contact conductivity detection and microchip electrophoresis was developed. Contact conductivity has traditionally been limited by the interaction of the separation voltage with the detection electrodes because the applied field creates a voltage difference between the electrodes, leading to unwanted electrochemical reactions. To minimize the voltage drop between the conductivity electrodes and therefore improve compatibility, a novel bubble cell detection zone was designed. The bubble cell permitted higher separation field strengths (600 V/cm) and reduced background noise by minimizing unwanted electrochemical reactions. The impact of the bubble cell on separation efficiency was measured by imaging fluorescein during electrophoresis. A bubble cell four times as wide as the separation channel led to a decrease of only 3% in separation efficiency at the point of detection. Increasing the bubble cell width caused larger decreases in separation efficiency, and a 4-fold expansion provided the best compromise between loss of separation efficiency and maintaining higher field strengths. A commercial chromatography conductivity detector (Dionex CD20) was used to evaluate the performance of contact conductivity detection with the bubble cell. Mass detection limits (S/N = 3) were as low as 89 +/- 9 amol, providing concentration detection limits as low as 71 +/- 7 nM with gated injection. The linear range was measured to be greater than 2 orders of magnitude, from 1.3 to 600 microM for sulfamate. The bubble cell improves the compatibility and applicability of contact conductivity detection in microchip electrophoresis, and similar designs may have broader application in electrochemical detection as the expanded detection zone provides increased electrode surface area and reduced analyte velocity in addition to the reduction of separation field effects.     

Dual conductivity/amperometric detection system for microchip capillary electrophoresis

The performance characteristics and advantages of a new dual electrochemical microchip detection system based on simultaneous conductivity and amperometric measurements are described. The system relies on the combination of a contactless conductivity detector with an end-column thick-film amperometric detector. Such coupling of the conductivity and amperometric detection modes in a single separation channel greatly enhances the sample characterization to offer simultaneous measurements of both ionic and electroactive species, improved reproducibility, and confirmation of peak identity. The simultaneous measurement of nitroaromatic and ionic explosives is used for demonstrating the ability to detect both electroactive and ionic species. Major improvements are also observed for analytes responding at both detectors. For example, the generation of dual response ratios can be used to improve the reproducibility and confirm the peak identity/integrity. Such dual response ratios reflect the distinct redox and conductivity properties of the individual analytes. The independence of the two detectors is reflected in the absence of "cross-talk" effects. The behavior of the dual detector is comparable with those of the individual detectors. Such a dual electrochemical detection system is easy to implement and requires inherently portable low-cost instrumentation.     

Contactless conductivity detection for capillary electrophoresis

A contactless capacitively coupled conductivity detector for capillary electrophoresis is introduced. The detector consists of two electrodes which are placed cylindrically around the outer polyimide coating of the fused-silica capillary with a detection gap of 2 mm. The electrodes form a cylindrical capacitor, and the electric conductivity of the solution in the gap between the electrodes is measured. A high audio or low ultrasonic frequency for coupling of the ac voltage is used in order to minimize the influence of reactance of the liquid. For an improved version of the detector, two syringe cannulas are used as the electrodes and the capillary is simply assembled into the tubing. This allows an easy placement of the detector on various positions along the capillary. The limit of detection of inorganic cations and anions is 200 ppb, as determined for sodium and chloride, respectively.     

On-chip contactless four-electrode conductivity detection for capillary electrophoresis devices

In this contribution, a capillary electrophoresis microdevice with an integrated on-chip contactless four-electrode conductivity detector is presented. A 6-cm-long, 70-microm-wide, and 20-microm-deep channel was etched in a glass substrate that was bonded to a second glass substrate in order to form a sealed channel. Four contactless electrodes (metal electrodes covered by 30-nm silicon carbide) were deposited and patterned on the second glass substrate for on-chip conductivity detection. Contactless conductivity detection was performed in either a two- or a four-electrode configuration. Experimental results confirmed the improved characteristics of the four-electrode configuration over the classical two-electrode detection setup. The four-electrode configuration allows for sensitive detection for varying carrier-electrolyte background conductivity without the need for adjustment of the measurement frequency. Reproducible electrophoretic separations of three inorganic cations (K+, Na+, Li+) and six organic acids are presented. Detection as low as 5 microM for potassium was demonstrated.     

Reduction of the impedance of a contactless conductivity detector for microchip capillary electrophoresis: compensation of the electrode impedance by addition of a series inductance from a piezoelectric quartz crystal

A low-impedance capacitively coupled contactless conductivity detector (LIC (4)D) for microchip capillary electrophoresis was reported. The LIC (4)D was the series combination of a piezoelectric quartz crystal (PQC) resonator with a capacitively coupled contactless conductivity detector (C (4)D) outside on the microchip lid. The electrode impedance in the LIC (4)D was reduced because the capacitive impedance from the wall capacitance was compensated by the inductive impedance from the PQC. The operation frequency of the LIC (4)D was set at the resonant frequency of the series combination of a PQC with a C (4)D, wherein a minimum in the total impedance was obtained. It was shown that the sensitivity of LIC (4)D was much higher than that of C (4)D itself, especially in the microchip with a thick lid. Under the experimental conditions, the signal-to-noise ratios of the LIC (4)D were improved by approximately 20-50 times over those of the C (4)D. Reproducible separations of a mixture of inorganic cations (K (+), Na (+), Li (+)) were demonstrated. After a digital filter treatment by the fast Fourier transform algorithm, the detection limits were 0.38, 0.49, and 1.6 microM for K (+) in the LI C (4)D with the microchip lid thickness of 0.20, 0.40, and 1.0 mm, respectively.     

Isoelectric focusing in a microfluidically defined electrophoresis channel

A new form of microchip isoelectric focusing that allows efficient coupling with pretreatment processes is reported. The sample is conveyed in a carrier ampholyte solution to the separation channel that is connected at both ends by two V-shaped lead channels, which supply electrode solutions to the connection point and complete the electrical connection to off-chip electrodes. The relatively high electric conductivity of the electrode solutions compared with that of the pH gradient enables focusing with a 2% loss of applied voltage at the electrodes using the lead channels. A glass microchip was constructed specifically for this configuration. The channel wall was coated with polydimethylacrylamide, and the IEF chip was operated in a chip holder equipped with on-chip connector valves. A plug of fluorescence-labeled peptide p I markers with p I values ranging from 3.64 to 9.56 with carrier ampholyte solution (pH 3-10) was introduced into the separation channel. When the plug reached the channel segment (24 mm in length) between the connection points with the electrolyte lead channels, isoelectric focusing was started after filling the lead channels with electrolyte solutions. The peptide markers were observed using scanning fluorescence detection. The entire range of the pH gradient was established in the segment after approximately 2 min. Isoelectric focusing of three consecutively injected sample plugs containing different p I markers was demonstrated.     

Flow-through sampling for electrophoresis-based microchips and their applications for protein analysis

This work presents a model behind the operation of a flow-through sampling chip and its application for immunoseparation, as well as its integration with a wash/elution bed for protein purification, concentration, and detection. This device used hydrodynamic pressure to drive the sample flow, and a gating voltage was applied to the electrophoretic channel on the microchip to control the sample loading for the separation and to inhibit sample leakage. The deduced model indicates that the critical gating voltage (VC) that is defined as the minimum gating voltage applied to the microchip for sampling is a function of the pump flow rate, the configuration of the microchannel on the chip, and the electroosmosis of the buffer solution. It was found that the theoretical V(C) values calculated from the measured electroosmotic mobilities and flow split ratios were comparable to those experimentally obtained from two microchips with different sampling channel sizes. This had an error percentage ranging from 1 to 20%. Because the hydrodynamic flow is insensitive to electrophoretic mobility, this electrophoresis-based microchip device was free of injection bias due to different ionic strength and electrophoretic mobility in the sample. Additionally, the usefulness of this device was demonstrated for the study of affinity interactions. Mixtures of Cy5-labeled bovine serum albumin (Cy5-BSA) and anti-BSA in various proportions were introduced into the microchip via a syringe pump, and the immunocomplex was electrophoretically separated from the free Cy5-BSA on the microchip. Based on the relative intensity of the free and complex BSA, the binding constant of BSA and anti-BSA was estimated as 3.3 x 10(7) M(-1). Furthermore, a C18 microcartridge (20 microL) was connected to the hydrodynamic inlet of the microchip. Using this device, the wash/elution step can be integrated on-line with the electrophoretic separation and detection on the microchip. Results show that the calibration curve of Cy5-BSA obtained from this integrated device has an R2 value greater than 0.99 and a minimum of quantitation at approximately 10 ng. This direct sampling method is another means of subfractionation, resulting in a relatively greater concentration factor than the average concentration of the whole fraction. Moreover, the electrical field-free bed ensures that the protein interaction will not be affected by the electric field during the wash/elution step.     

低电压电泳芯片及其在生化样品分析中的应用

为了解决微流控电泳芯片集成化问题,设计并制作出一种具有管道两侧微阵列电极结构的硅-PDMS复合低电压电泳芯片.通过电路控制程序在微侧壁阵列电极上施加交替循环的低电压,以实现芯片微管道中低电压电泳过程;并对硅-PDMS芯片的电绝缘性、伏安曲线及电渗流等性能进行了测试和评价.以pH为10.0、10mmol/L的硼砂作为缓冲体系,分离场强150V/cm、切换时间3s的条件下,完成了10-4mol/L的苯丙氨酸和精氨酸的低电压电泳分离,分离度达1.6,实现了两种氨基酸的完全分离.在此基础上,将系统用于牛血清白蛋白和α-乳白蛋白的分离,并初步实现了该两种蛋白质的芯片电泳分离.     

Microchip capillary electrophoresis with an integrated indium tin oxide electrode-based electrochemiluminescence detector

This paper describes an indium tin oxide (ITO) electrode-based Ru(bpy)3(2+) electrochemiluminecence (ECL) detector for a microchip capillary electrophoresis (CE). The microchip CE-ECL system described in this article consists of a poly(dimethylsiloxane) (PDMS) layer containing separation and injection channels and an electrode plate with an ITO electrode fabricated by a photolithographic method. The PDMS layer was reversibly bound to the ITO electrode plate, which greatly simplified the alignment of the separation channel with the working electrode and enhanced the photon-capturing efficiency. In our study, the high separation electric field had no significant influence on the ECL detector, and decouplers for isolating the separation electric field were not needed in the microchip CE-ECL system. The ITO electrodes employed in the experiments displayed good durability and stability in the analytical procedures. Proline was selected to perform the microchip device with a limit of detection of 1.2 microM (S/N = 3) and a linear range from 5 to 600 microM.     

Integrated light collimating system for extended optical-path-length absorbance detection in microchip-based capillary electrophoresis

We have developed an integrated light collimating system with a microlens and a pair of slits for extended optical path length absorbance detection in a capillary electrophoresis (CE) microchip. The collimating system is made of the same material as the chip, poly(dimethylsiloxane) (PDMS), and it is integrated into the chip during the molding of the CE microchannels. In this microchip, the centers of an extended 500-microm detection cell and two optical fibers are self-aligned, and a planoconvex microlens (r = 50 microm) for light collimation is placed in front of a light-delivering fiber. To block stray light, two rectangular apertures, realized by a specially designed three-dimensional microchannel, are made on each end of the detection cell. In comparison to conventional extended detection cell having no collimator, the percentage of stray radiation readout fraction in the collimator integrated detection cell is significantly reduced from 31.6 to 3.8%. The effective optical path length is increased from 324 to 460 microm in the collimator integrated detection cell. The detection sensitivity is increased by 10 times in the newly developed absorbance detection cell as compared to an unextended, 50-microm-long detection cell. The concentration detection limit (S/N = 3) for fluorescein in the collimator integrated detection cell is 1.2 microM at the absorbance detection limit of 0.001 AU.     

Detection of human immunoglobulin in microchip and conventional capillary electrophoresis with contactless conductivity measurements

The detection of human immunoglobulin M (IgM) was performed using capacitively coupled contactless conductivity detection (CCD) in electrophoresis carried out in conventional capillaries as well as on glass and poly(meth-yl methacrylate) (PMMA) microdevices. Also achieved was the analyses of IgG (an anti-human IgM) and the complex formed in the reaction between the two immunoreagents. It is demonstrated that CCD is a powerful tool suitable not only for the detection of antibodies but also for monitoring an immunological interaction. Conductivity measurements allow the direct determination of immunoreagents, and it is advantageous, since no labels are required. The immunoglobulin IgM has been taken as model analyte. The reproducibility of the analytical signal (RSD = 1%), sensitivity and limits of detection obtained for IgM (0.15 ng/mL in conventional capillaries and 34 ng/mL in microchips) are comparable to those previously obtained with amperometric detection. The immunological reaction was performed either in conventional microtiter plates as used in ELISA or in situ on the glass chip.     

Microchip capillary electrophoresis with solid-state electrochemiluminescence detector

We report microchip capillary electrophoresis (CE) coupling to a solid-state electrochemiluminescence (ECL) detector. The solid-state ECL detector was fabricated by immobilizing tris(2,2'-bipyridyl)ruthenium(II) (TBR) into an Eastman AQ55D-silica-carbon nanotube composite thin film on an indium tin oxide (ITO) electrode. After being made by a photolithographic method, the surface of the ITO electrode was coated with a thin composite film through a micromolding in capillary (MIMIC) technique using a poly(dimethylsiloxane) (PDMS) microchannel with the same pattern as an ITO electrode. Then the TBR was immobilized via ion exchange by immersing the ITO electrode containing the thin film in TBR aqueous solution. The whole system was built by reversibly sealing the TBR-modified ITO electrode plate with a PDMS layer containing electrophoresis microchannels. The results indicated that the present solid-state ECL detector displayed good durability and stability in the microchip CE-ECL system. Proline was selected to perform the microchip device with a limit of detection of 2 microM (S/N=3) and a linear range from 25 to 1000 microM. Compared with the CE-ECL of TBR in aqueous solution, while the CE microchip with solid-state ECL detector system gave the same sensitivity of analysis, a much lower TBR consumption and a high integration of the whole system were obtained. The present system was also used for medicine analysis.